Chromatography is a well-established and valuable technique for separating chemical and biological substances and is widely used in research and industry, finding many applications in compound preparation, purification and analysis. There are many different forms of chromatography, liquid chromatography being of particular importance in the pharmaceutical and biological industries for the preparation, purification and analysis of proteins, peptides and nucleic acids.
A typical liquid chromatography apparatus has an upright housing in which a bed of packing material, which is usually particulate in nature and consists of a porous medium, rests against a permeable retaining layer. A liquid mobile phase enters through an inlet, for example at the top of the column, usually through a porous, perforated filter, mesh or frit, moves through the bed of packing material and is removed via an outlet, typically through a second filter, mesh or frit.
Columns used in liquid chromatography typically comprise a tubular body enclosing the porous chromatography medium through which the carrier liquid or mobile phase flows, with separation of substances or analytes taking place between the mobile phase and solid phase of the porous medium. Typically, the porous medium is enclosed in the column as a packed bed, generally formed by consolidating a suspension of discrete particles, known as slurry that is pumped, poured or sucked into the column, usually from a central bore or nozzle located at one end of the column. The production of a stable, even bed is critical to the final separation process and optimum results are found using bores which are centrally positioned through at least one column end piece.
Another critical feature in the separation of substances is the fluid distribution system, particularly as the cross-section of the chromatographic column increases. The efficiency of the chromatographic separation relies on the liquid distribution and collection system at the fluid inlet and outlet of the packed bed.
Ideally, the carrier liquid is uniformly introduced throughout the surface at the top of the packing, flows through the packing at the same velocity throughout the packing cross section, and is uniformly removed at the plane defined by the bottom of the packed bed.
Conventional distribution systems for use in liquid chromatography must address a number of inherent problems that have deleterious effects on the separation efficiency of the column. Among these problems is the non-uniform initial fluid distribution at the top of the packed bed. The problem of non-uniform initial fluid distribution refers generally to the problem of applying a sample volume simultaneously over the cross-sectional area of the packed bed. This problem will lead to increased dispersion in the chromatographic system by broadening the convective residence time distribution of a tracer substance transported with the fluid throughout the system. The dispersion generated by the liquid distribution system has to be controlled in relation to the amount of dispersion introduced by the chromatographic packed bed itself by means of diffusion and mixing effects. Without a simultaneous introduction of fluid in the plane defined by the top of the bed, it is virtually impossible to achieve so-called plug-flow behaviour, which is a uniform and well-defined movement of the sample through the packed bed and column, respectively, resulting in a uniform residence time distribution.
Standard fluid distribution systems consist of one central inlet for the mobile phase in combination with a thin distribution channel (gap) adjacent to the particle retaining filter (mesh, woven net or sinter) confining the top and bottom plane of the inlet and outlet of the packed bed. In theory and from experience it is known that such a system deteriorates in performance with increasing diameter of the column. This is due to the residence time difference between fluid elements travelling from the inlet to the outer column wall and those fluid elements which directly can enter the filter or net and the packed bed region below the inlet port. This difference in residence time is enlarged with column diameter and leads to chromatographic band broadening which becomes most severe with small particles. This problem corresponds to the non-uniform initial fluid distribution.
Non-uniform fluid distribution across the surface of the packed bed is also emanating from a reduction in the filter area in contact with the packed bed surface. For example, when providing a medium inlet, here also called a nozzle, for introducing the media into the column, which nozzle is protruding into the column centrally in one of the end units through the distribution system and the filter, the mobile phase can apparently not be added to the column exactly here as a central portion of the filter area is taken by the medium inlet. It is therefore desirable to reduce the size of this medium inlet as much as possible in order to maintain a large filter area and thereby minimizing the distortion in the flow pattern as much as possible. In practice, it is not only the size of the nozzle as such that causes a reduction in accessible area for fluid distribution across the packed bed, but also the sealing means around the nozzle. In order to avoid leakage of resin (particulate media) into the mobile phase a tight seal around the nozzle is required. Furthermore, sufficient mechanical support has to be provided such that the filter is kept in place around the nozzle. One way to do this is by welding the filter against a so-called nozzle retainer. However, welding is costly so other methods may be preferred. Another possibility is to provide a filter holder below the filter and around a part of the nozzle to prevent leakage. The filter holder comprises one squeezing means, here in the form of a cylindrical part which is for example a threaded cylinder adapted to surround the lowest part of the nozzle and one filter squeezing part for example in the form of a plate with a central hole adapted to receive the nozzle. Said filter squeezing part is attached to the cylindrical part such that the nozzle can pass through. The filter squeezing part is adapted to squeeze the filter against a corresponding element at the end piece of the column in order to prevent leakage of resin into the mobile phase in between the filter squeezing part and the filter. In order to get a reliable leak protection the area of the filter squeezing part needs to be a bit larger than a medium inlet passage provided in the filter for letting the medium inlet (nozzle) through. This will lead to a larger area in the column centreline blocking the liquid flow towards the packed bed and increasing the problem of non-uniform initial fluid distribution over the packed bed as described above.
For small columns this area of the filter squeezing part compared to the total cross section area of the column is relatively large and since no mobile phase will be applied beneath this filter holder area the mobile phase distribution is not optimal and the column efficiency will be reduced when operating the column. Furthermore, during sanitization of the packed bed it is important that the sanitization agents will reach the total volume of the packed bed efficiently which will be compromised when a large filter holder is blocking a too large area of the packed bed surface.